Western blues

David Boucher David.Boucher at med.monash.edu.au
Tue Sep 26 00:27:35 EST 2000


Paul your mum just rang and says you forgot your lunch again

Paul Cullen wrote:
> 
> Are you pre-wetting the membrane with methanol before assembling the
> transfer apparatus? I've found this to be necessary with Immobilon P.
> 
> deepika wrote:
> >
> > Hi
> > I have been having a lot of problems with efficiency of transfer from
> > gel (10% acrylamide/bis) to PVDF membrane (Immobilon P). When I stain
> > the gel after transfer, there is a lot of protein left on there. I know
> > that I am not exceeding the binding capacity of the membrane (I did a
> > titration of ug protein loaded and discovered that the ratio of protein
> > transferred to those that stay behind is the same).
> >             The protein of interest is high moleculat weight (around 160
> > kDa). I have tried :
> > - reducing MeOH concetration in the buffer from 20% to 10 %.
> > - doing an O/N transfer (25 V) followed by 1/2 hr 100 V
> > - doing a 2 hr 100 V transfer
> >
> > Nothig really helps. Somethimes there is enough protein for me to see (I
> > use LumiLight, Roche) and sometimes there's not. So help, please !! Are
> > high mol wt proteins transferred better at high or low field strength ?
> > Has anyone tried SDS in the transfer buffer ?
> >
> > Thanks
> > Deepika
> >
> > ---
> 
> --
> Paul Cullen
> Bacterial Pathogenesis Research Group
> Monash University
> Australia
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